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Structural and Functional Characterization of Malate Synthase G from Opportunistic Pathogen Pseudomonas aeruginosa Alyssa C. McVey,† Prasanthi Medarametla,‡ Xavier Chee,§ Sean Bartlett,∥ Antti Poso,‡,⊥ David R. Spring,∥ Taufiq Rahman,§ and Martin Welch*,† †

Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, U.K. School of Pharmacy, University of Eastern Finland, 70211 Kuopio, Finland § Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, U.K. ∥ Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K. ⊥ Department of Internal Medicine VIII, University Hospital Tübingen, 72076 Tübingen, Germany ‡

S Supporting Information *

ABSTRACT: Pseudomonas aeruginosa is an opportunistic human pathogen recognized as a critical threat by the World Health Organization because of the dwindling number of effective therapies available to treat infections. Over the past decade, it has become apparent that the glyoxylate shunt plays a vital role in sustaining P. aeruginosa during infection scenarios. The glyoxylate shunt comprises two enzymes: isocitrate lyase and malate synthase isoform G. Inactivation of these enzymes has been reported to abolish the ability of P. aeruginosa to establish infection in a mammalian model system, yet we still lack the structural information to support drug design efforts. In this work, we describe the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. The enzyme is a monomer composed of four domains and is highly conserved with homologues found in other clinically relevant microorganisms. It is also dependent on Mg2+ for catalysis. Metal ion binding led to a change in the intrinsic fluorescence of the protein, allowing us to quantitate its affinity for Mg2+. We also identified putative drug binding sites in malate synthase G using computational analysis and, because of the high resolution of the experimental data, were further able to characterize its hydration properties. Our data reveal two promising binding pockets in malate synthase G that may be exploited for drug design.

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of hospital infections.6−8 These infections escalate the need for surgical intervention, increase the overall length and costs of hospitalization, and can carry an average mortality rate of 60%.9 PA is also associated with certain types of chronic infection, most notably in the airways of patients with cystic fibrosis.10 These infections are often especially difficult to resolve because of the high acquired and intrinsic antibiotic resistance of PA. Certainly, the lack of new anti-Pseudomonal drugs available to the clinical community recently led the World Health Organization to classify PA as a “Priority 1: Critical” pathogen for the development of new antibiotics.11 Expression levels of the glyoxylate shunt enzymes are upregulated in chronic PA infections of cystic fibrosis patients.12,13 Additionally, ICL and MS appear to be vital for fitness of PA in mammalian infection models. An ICL deletion mutant displayed an increased rate of clearance from the lung and a reduced level of tissue damage in a rat pulmonary infection model.2,14 Likewise, a PA double mutant (ΔICL

alate synthase isoform G (MS) is the second enzyme of the glyoxylate shunt, which is an anaplerotic pathway that diverts carbon skeletons away from the catabolic reactions of the tricarboxylic acid cycle by providing a route from isocitrate directly to gluconeogenic precursors.1 The glyoxylate shunt is present in a wide range of prokaryotic and lower eukaryotic organisms1,2 and allows the synthesis of cell constituents from metabolic inputs containing (or metabolized to) a C2 backbone, such as fatty acids, acetate, or ethanol. Instead of undergoing oxidative decarboxylation to yield αketoglutarate, first isocitrate is cleaved to yield glyoxylate and succinate in a reversible reaction catalyzed by isocitrate lyase (ICL). Thereafter, MS catalyzes the second reaction in the glyoxylate shunt, which is the irreversible condensation of glyoxylate and acetyl-coenzyme A (AcCoA) to yield coenzyme A (CoA) and malate. The shunt enzymes have been implicated in virulence, persistence, and antibiotic resistance in several medically relevant organisms,2−5 including the opportunistic pathogen Pseudomonas aeruginosa (PA). PA is a Gram-negative bacillus that has been reported to occupy a variety of ecological niches.6 It is also a notorious nosocomial pathogen, accounting for 15% © XXXX American Chemical Society

Received: August 30, 2017 Revised: September 25, 2017

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DOI: 10.1021/acs.biochem.7b00852 Biochemistry XXXX, XXX, XXX−XXX

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dialyzed extensively at 4 °C in 50 mM Tris-HCl (pH 7.5) containing 100 mM NaCl and 10 mM EDTA. The EDTA was then removed by dialysis at 4 °C against 3 × 1 L of 50 mM Tris-HCl (pH 7.5) containing 100 mM NaCl. The final protein concentration was adjusted to 175 μM after dialysis. Kinetic Measurements of MS. The enzymatic activity of MS was measured using a modified coupled assay in which the amount of free thiol (present in the reaction product, CoA) was titrated using 5,5′-dithio(2-nitrobenzoic acid) (DTNB).19 Briefly, reaction mixtures contained buffer [50 mM dipotassium phosphate and 15 mM MgCl2 (pH 7.5)] supplemented with AcCoA and glyoxylate (as indicated) and 0.2 mM DTNB. The mixture was equilibrated at 37 °C for 1 min before the reaction was initiated by addition of MS (final concentration of 25 nM). The initial linear change in absorbance (ΔAbs) was recorded at 412 nm using a Biospectrometer (Eppendorf). Rates were calculated using the molar extinction coefficient of DTNB (14150 M−1 cm−1). Kinetic parameters were calculated from best-fit nonlinear regression using GraphPad Prism version 6. Crystallization of MS. Crystallization conditions were screened using sitting drop vapor diffusion with ≥11 mg/mL purified protein. The best crystals were obtained with a 1:1 mixture of protein and reservoir solution [0−25 mM ammonium sulfate, 13−17% (v/v) glycerol, and 20−27% (w/ v) PEG 4000] at 19 °C. Crystals were grown for 2−6 days, treated with a cryoprotectant [24% (v/v) ethylene glycol and 76% (v/v) mother liquor], mounted in nylon loops, and flashfrozen in liquid nitrogen prior to data collection. X-ray Diffraction, Structure Determination, and Refinement. Diffraction data were collected remotely on beamline MX-I03 at the Diamond Light Source Synchrotron (DLS, Didcot, U.K.). Diffraction data were processed using Xia3 DIALS, and the structure was determined by molecular replacement using Phaser.20 We used the apo MS structure from Mtb (PDB entry 1N8I) as a model. Automated refinement was performed using PHENIX.refine.21 Manual modeling and refinement were performed in COOT.22 Data collection and refinement statistics are listed in Table 1. Fluorimetry. Fluorescence spectra were recorded on a PerkinElmer LS55 luminescence spectrometer using an optical cuvette with a 1 cm path length. Mg2+-depleted MS was added to a buffer composed of 50 mM Tris-HCl (pH 7.5) and 100 mM NaCl, giving a final MS concentration of 0.88 μM. MgCl2, glyoxylate, and AcCoA were added to give the final indicated concentrations. The mixtures were equilibrated at 25 °C for 2 min prior to measurement. An excitation wavelength of 295 nm was used for measurement of intrinsic tryptophan fluorescence, and the emission spectra were recorded between 300 and 400 nm. The excitation and emission slit widths were 2.5 nm. The data presented are the mean of three independent repeats. Computational Analysis of MS. The Schrödinger suite (Schrö dinger, LLC, New York, NY) was used for all computations. Protein Preparation Wizard with default parameters was used to prepare the MS structure for downstream in silico analyses.23 SiteMap was used to predict putative binding pockets. This algorithm predicts binding pockets in proteins on the basis of the geometry, size, volume, and nature (hydrophilicity/hydrophobicity) of amino acid residues.24 SiteMap predicts druggable pockets and ranks them using two parameters: the SiteScore and the druggability or “Dscore”. SiteScore reveals highly promising binding sites in the protein that can potentially be targeted with drugs, and Dscore indicates the druggability of the predicted site. SiteMap

ΔMS) was found to be completely avirulent in a mouse pulmonary infection model.15 Thus, both ICL and MS are attractive targets for drug discovery because they are essential during infection situations. Crucially, no orthologs of the shunt enzymes exist in humans.4 Examination of the crystal structures of shunt enzymes from Mycobacterium tuberculosis (Mtb) suggested that MS would provide a more druggable target than ICL would because of its deeper, more hydrophobic active site, which has to accommodate the pantothenate tail of AcCoA.16,17 Moreover, drugs that target the shunt enzymes have potential as broad spectrum antibiotics given their conservation across many pathogenic species.18 In this work, we present the 1.62 Å resolution structure of malate synthase G from P. aeruginosa. We then use this structural data to identify and describe the most suitable binding pockets for drug development in this critical priority pathogen. Furthermore, we use molecular dynamic simulation to yield the free energy of bound water molecules, mapping the likely physiological hydration network of the protein.

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EXPERIMENTAL PROCEDURES Cloning, Overexpression, and Purification. MS (GlcB, PA0482) from P. aeruginosa strain PAO1 was overexpressed with a Precission protease-cleavable, N-terminal GST tag. A 2 L culture of Escherichia coli strain BL21 containing plasmid [pGEX-6P-1 (glcB)]19 was grown with good aeration at 37 °C in 2×TY medium to an OD600 of 0.6. The temperature was then decreased to 16 °C; IPTG was added to a final concentration of 0.1 mM to induce expression of the cloned glcB ORF, and protein expression was continued for a further 16 h. The cells were harvested by centrifugation (3430g for 30 min at 4 °C), and the cell pellets were resuspended in 30 mL of ice-cold lysis buffer (phosphate-buffered saline supplemented with 1 mM DTT and an EDTA-free protease inhibitor cocktail tablet). The cells were lysed to completion by sonication on ice. Cell debris was removed by centrifugation (15000g for 30 min at 4 °C), and the supernatant was filtered through a 0.45 μm membrane filter. Subsequent purification was performed using an Ä KTA FPLC system. The filtered sample was loaded immediately onto a 5 mL glutathione sepharose column (GSTrapFF) equilibrated with buffer A [150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, and 0.1 mM TCEP (pH 7.5)]. The column was washed with buffer A, and the bound protein was eluted with buffer A supplemented with reduced glutathione (10 mM). Meanwhile, 5 mL of slurried glutathione Sepharose 4B resin (GE Healthcare) was equilibrated with buffer A in a 50 mL polypropylene tube. The GST-tagged MS fusion protein was added to the glutathione sepharose beads along with 50 units of PreScission Protease (GE Healthcare). The mixture was then incubated for 48 h at 4 °C while being gently mixed to allow cleavage of the fusion protein from the immobilized GST tag. The supernatant was loaded directly onto a 16-600 Superdex 200 preparatory grade column (GE Healthcare) equilibrated with buffer A. The flow rate was 0.8 mL min−1, and the eluate was collected in 2 mL fractions. Fractions containing purified MS were pooled, concentrated, frozen in liquid nitrogen, and stored at −80 °C. The protein concentration was determined by spectrophotometry using the calculated molar extinction coefficient for PA MS (εcalc = 75985 M−1 cm−1). Preparation of Mg2+-Depleted MS. After batch purification, 50 mM EDTA was added to a portion of purified MS to remove bound metals for fluorescence studies. MS was then B


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understand the druggability of MS through its solvation network.

Table 1. Summary of Data Collection and Refinement Statistics for P. aeruginosa Malate Synthase G

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Data Collectiona X-ray source wavelength (Å) resolution range (Å) total no. of reflections no. of unique reflections multiplicity completeness (%) mean I/σ(I) Rsym Rmeas CC1/2 space group unit cell a (Å) b (Å) c (Å) α = β = γ (deg) no. of molecules per asymmetric unit Refinement Rwork (%) Rfree (%) no. of atoms protein glycerol 1,2-ethanediol ions water root-mean-square deviation bond lengths (Å) bond angles (deg) Wilson B factor (Å2) average B factor (Å2) overall protein glycerol 1,2-ethanediol ions water Ramachandran analysis (%) favored allowed outliers rotamer outliers MolProbity Clashscore PDB entry a

RESULTS AND DISCUSSION Kinetic Analysis. Purified PA MS exhibited Michaelis− Menten kinetics for glyoxylate and AcCoA substrates (Figure 1). The relevant steady state kinetic parameters are listed in

DLS MX-I03 0.97625 71.17−1.62 556009 (9988) 96326 (3151) 5.8 (3.2) 94.5 (69.9) 11.75 (1.47) 0.066 (0.912) 0.079 (1.080) 1 (0.5) P212121 71.17 81.2 137.57 90 1 16.7 19.6 6350 4 15 2 691

Figure 1. Michaelis−Menten kinetics of P. aeruginosa malate synthase G. Enzyme activity was measured by fixing the concentration of one substrate [either AcCoA (A) or glyoxylate (B)] at 0.2 mM and varying the concentration of the second substrate as indicated. Each assay was performed in triplicate. Error bars correspond to ±1 standard deviation.

0.0008 0.897 19.6 27.30 25.49 56.28 60.92 54.90 38.76

Table 2. Comparison of Kinetic Parameters of MS from Other Microorganisms

98 1.9 0.1 0.3 5 5OAS

Values for the highest-resolution shell are given in parentheses.

was run using a fine grid option with a more restrictive definition of hydrophobicity to exclude solvent-exposed regions during binding site prediction. The solvation network plays an important role in determining protein−ligand binding affinity and may be useful when designing or conducting virtual screening of potential inhibitors. Probable hydration sites and associated thermodynamic properties were predicted using the WaterMap module.25 WaterMap predicts hydration sites and their properties in user-defined regions on the basis of molecular dynamics. WaterMap was run using default parameters to

organism

Km,gly (μM)

Km,AcCoA (μM)

Vmax (μmol mg−1 s−1)

ref

P. aeruginosa P. aeruginosa M. tuberculosis M. tuberculosis E. coli C. glutamicum

34 70 30 57 21 30

47 12 10 30 9 12

1200 990 23 (kcat) 360 2160 −

this work 19 28 26 27 29

Table 2. Kinetic analysis of purified PA MS has been performed in just one study, which reported a lower substrate affinity for glyoxylate and a higher substrate affinity for AcCoA.19 Results from kinetic analyses of other MSs from Mtb, E. coli, and Corynebacterium glutamicum were comparable (Table 2).26−28 Additionally, Roucourt et al.19 have determined that the PA MS enzymatic reaction proceeds through a sequential mechanism, as opposed to a substituted mechanism. Amino Acid Sequence Alignment. We aligned MS amino acid sequences sourced from the Uniprot database (P. aeruginosa, Q9I636; E. coli, P37330; M. tuberculosis, P9WK17; C


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Figure 2. Amino acid sequence alignment of P. aeruginosa malate synthase G with homologues whose structures have been determined by X-ray crystallography: E. coli, P37330, PDB entry 1P7T; M. tuberculosis, P9WK17, PDB entry 1N8I; M. leprae, O32913, PDB entry 4EX4. Amino acids are colored by sequence identity, where regions of 100% sequence identity are highlighted in purple. Amino acids known to be involved in catalysis in E. coli and M. tuberculosis are highlighted in orange; these are conserved among all four organisms shown.

Mycobacterium leprae, O32913) with Clustal Omega30,31 and displayed the alignment with Jalview32 (Figure 2). There is 60% sequence identity between PA and E. coli MS and 70% sequence identity between PA and Mtb MS. The amino acid residues that are identical among all four species are highlighted in purple, and the residues known to be involved in catalysis in Mtb and E. coli are highlighted in orange.26−28,33 We also compared amino acid identity within a larger group of pathogenic organisms that encode MS, signifying the evolutionary relationship of MS in other medically relevant organisms (Figures S1 and S2). Tertiary Structure of MS. We crystallized PA MS and determined the three-dimensional structure by molecular replacement using Mtb MS (PDB entry 1N8I) as a template. PA MS crystallized with one molecule in the asymmetric unit. Consistent with this, MS also behaved as a monomer in solution (Figure S3). The model (Figure 3) was optimized with an Rwork of 16.7% and an Rfree of 19.6%. The Ramachandran distribution of all amino acid residue dihedral angles in the protein structure indicates that 98% of the amino acids are in favored or allowed regions (Figure S4). The structure of MS in the apo, substrate-bound, or product-bound form from three

Figure 3. Cartoon representation of malate synthase G from P. aeruginosa (PDB entry 5OAS). The TIM barrel, α-helical, and β-sheetrich domains are colored blue, green, and red, respectively. The Nterminus (amino acids 2−115) is colored light cyan. A closer look at the active site shows the proposed substrate binding residues, Glu432 and Asp460, depicted as magenta sticks and the proposed catalytic residues, Asp631 and Arg340, shown as orange sticks. The residues involved in catalysis are oriented around a Mg2+ ion, which is depicted as a chartreuse sphere.

other prokaryotic species has been elucidated by X-ray crystallography: E. coli (PDB entries 1P7T and 2JQX), Mtb (PDB entries 1N8W, 1N8I, and 2GQ3), and M. leprae (PDB D


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Biochemistry entry 4EX4). In addition, while our manuscript was in preparation, we became aware that a structure of P. aeruginosa MS with glyoxylate bound in the active site had been deposited in the PDB (entry 5VFB). However, to the best of our knowledge, this structure has not yet been described in the literature. In common with these previously reported MS structures, PA MS is composed of three domains: a TIM barrel domain with eight α-helices and eight β-strands (amino acids 116−135 and 266−589), a helical domain (amino acids 590− 725), and a β-sheet-rich domain (amino acids 136−265) inserted between the first α-helix and second β-sheet of the TIM barrel domain. Near the N-terminus of the protein is a strikingly long α-helix (amino acids 32−71) that partially wraps around the TIM barrel domain (Figure 3). Comparison of the structure presented here with known structures of MS from other organisms revealed that the active site residues and the Mg2+ ion are highly conserved across species.26,27,33−37 For example, the backbone rmsd of the superposition between the Mtb MS used as the molecular replacement template (PDB entry 1N8I) and PA MS is 0.94 Å over 682 Cα atoms, and among the active site residues, the rmsd is 0.20 Å. The active site sits between the TIM barrel and a loop (amino acids 615−631 in PA) from domain II, and the known active site residues are present in the PA MS structure in the same positions (not shown). The active sites compared between apo and substrate-bound crystal structures of MS are practically identical, except for the tightening of the aforementioned loop from domain II when glyoxylate is bound (not shown), so MS is generally not considered to undergo major conformational changes during catalysis.35 Additionally, comparison of the apo structure presented here with the glyoxylate-bound structure of PA MS recently deposited in the PDB (entry 5VFB) revealed no conformational differences except for a small shift in the solvent-exposed loop (amino acids 305−312) far from the active site. Influence of Mg2+ on Substrate Binding and the Active Site Environment. The PA MS crystal structure revealed electron density in the F0 − Fc difference map for a Mg2+ ion and a molecule of ethylene glycol (EDO), the cryoprotectant used when mounting MS crystals, in the active site. Mg2+ is reportedly essential for the enzymatic function of MS and is present in every MS crystal structure published. In these other structures, the Mg2+ is coordinated by four water molecules and conserved Glu and Asp residues in an octahedral conformation.26,27,35,36,38,39 However, in our structure, the Mg2+ appears to be coordinated by the EDO molecule, one water molecule, and the side chains of Glu432, Asp460, Glu274, and Asp275 (Figure 4). These amino acids contain carboxylate groups, all located within 3.5 Å of the metal ion, and could be interacting to decrease the overall energy. We also observed a nearby conserved tryptophan residue (Trp539 in our structure), which could be used as a fluorescent reporter to monitor metal ion binding in the active site. To test this, we measured the intrinsic tryptophan fluorescence of PA MS to quantify the binding affinity of Mg2+ and to examine the effect of the substrate(s) on Mg2+ binding. To investigate the Mg2+ binding properties of PA MS, we first removed any prebound Mg2+ from the purified protein. We observed a change in relative tryptophan fluorescence intensity and λmax upon addition of Mg2+ and substrates to demetallized MS. We determined λmax for demetallized MS as 340 nm, which was red-shifted to 342 nm upon addition of 10 mM MgCl2 (Figure 5A). This indicates that the environment around the

Figure 4. Mg2+ is coordinated by Glu274, Asp275, Glu432, Asp460, ethylene glycol (EDO), and a single water molecule in the active site of P. aeruginosa malate synthase G. The electron density map is contoured at 1.5σ. Trp539 is located ∼6 Å from the Mg2+ in the crystal structure (PDB entry 5OAS).

Figure 5. (A) Fluorescence spectra of demetallized malate synthase G (black line), demetallized malate synthase G with 10 mM Mg2+ added (blue dashes), and demetallized malate synthase G with 10 mM Mg2+ and 1 mM glyoxylate added (red dots). The λmax for each spectrum is indicated by a vertical dashed line of the corresponding color. (B) Stern−Volmer analyses indicate no global conformational changes as a result of binding of Mg2+ or glyoxylate to malate synthase G. Error bars correspond to ±1 standard deviation.

tryptophan residue(s) becomes more hydrophilic and solventexposed in the presence of Mg2+. Conversely, λmax was blueshifted to 337 nm after the addition of 1 mM glyoxylate (in the presence of 10 mM MgCl2), indicating that the environment around Trp539 becomes more hydrophobic and less exposed to the bulk solvent. However, when 1 mM glyoxylate was added to demetallized MS in the absence of added Mg2+, there was no deviation in λmax or fluorescence intensity compared with the spectrum of the demetallized protein alone, implying that Mg2+ is needed in the active site for glyoxylate to bind (Figure S5A). Interestingly, a comparison of the structure described here with that of PA MS with bound glyoxylate (PDB entry 5VFB) E


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presence of glyoxylate, respectively (Figure 6B). This is comparable with the binding affinity of Mg2+ for other enzymes.41 On the basis of sequence and structural similarity, we propose that the amino acid residues involved in catalysis in PA MS are Arg340, Glu432, Asp460, and Asp631 (Figures 2 and 3). We propose a mechanism, consistent with our fluorescence data and previous studies on MS in Mtb and E. coli,26−28,33 in which Mg2+ is coordinated in an octahedral conformation by the carboxylate side chains of Glu432 and Asp460, along with four water molecules.39 In this model (Figure S7), glyoxylate enters the active site and displaces two of the water molecules in the Mg2+ coordination sphere. Following this, AcCoA enters the active site. Asp631 then deprotonates AcCoA.38 The resulting enolate attacks glyoxylate (activated by chelation of Mg2+) to form a malyl-CoA intermediate that, in turn, deprotonates a proximal water molecule. This affords a hydroxide anion that can attack and hydrolyze the thioester. Arg340 then acts as a catalytic acid, protonating CoA, which causes CoA to leave the active site first, followed by malate.28 Identification of Potential Binding Sites in PA MS. Because PA is such a high-priority pathogen for new antibiotic discovery efforts, we wanted to identify any potential binding sites in PA MS that might facilitate structure-based drug design. Therefore, we analyzed the PA MS crystal structure using Schrödinger’s SiteMap. We denoted the five top-ranking potential binding sites identified by SiteMap as A−E, and their SiteMap parameters are listed in Table 3. The location of each site on the crystal structure is shown in Figure 7A as colored mesh (site A, yellow; site B, orange; site C, magenta; site D, blue; site E, cyan). Sites A−E possess a SiteScore of >0.80, which is the minimum recommended value to distinguish between a drug binding and non-drug binding site.24 Site A is the top-ranked potential binding site with a SiteScore of 1.10 and a Dscore of 1.16. Site B is second-ranked with a SiteScore of 0.99 and a Dscore of 1.02. These scores are consistent with those from earlier studies in proteins in which drugs that bind with submicromolar affinity have been identified,24 indicating that sites A and B are promising binding sites that could be used for structure-driven drug design. Site A (Figure 7B) is narrow and highly hydrophobic in nature, with a hydrophobic-to-hydrophilic character ratio of 2.22. For reference, the mean hydrophobicto-hydrophilic ratio for tight binding sites in proteins is 1.60.24 This ratio, along with the SiteScore and Dscores calculated by SiteMap, implies that this pocket is suitable for exploration using druglike molecules. Also, because the pocket is located deep within the protein, small molecules could be accommodated, rather than larger ones, whose entry may be hindered. Site B has the largest predicted volume (569 Å3) and spreads over the entrance of the active site. Site B has a hydrophobic interior region and a hydrophilic exterior, with a hydrophobicto-hydrophilic character ratio of 0.61. The residues in site B are displayed in Figure 7C. Having identified potential ligand binding sites in PA MS, we assessed the likely impact on enzyme function of binding of the drug to these sites, and their degree of conservation across species (including Mtb, PDB entry 5CC5; E. coli, PDB entry 1P7T; and M. leprae, PDB entry 4EX4). The protein structure alignment revealed that site B incorporates the known substrate binding sites of MS (Figure 8) and contains two of the residues involved in catalysis in PA, Arg340 and Aps631, and the active site fluorophore, Trp539 (Figure 7C). Site A also partially

reveals no apparent conformational differences between the active sites. Together with the results outlined here, this is consistent with physical occlusion of the bulk solvent in the active site by the substrate. To establish whether binding of Mg2+ or glyoxylate to PA MS is accompanied by global conformational changes, we measured the accessibility of the tryptophan residues to an extrinsic neutral quencher, acrylamide. The presence of increasing concentrations of acrylamide caused progressively stronger quenching of tryptophan fluorescence, but the apparent dynamic quenching constant (KSV = 10.5 ± 0.2 M−1) was similar for demetallized MS and for Mg2+-bound or glyoxylate-bound MS (Figure 5B). This indicates that the changes in tryptophan fluorescence arising from Mg2+ or glyoxylate binding arise as a consequence of changes in the local microenvironment of one or more Trp residues, most likely Trp539, in the active site. We also examined whether the secondary structure content of the protein was altered in the presence of its substrates using circular dichroism (Figure S6). In contrast to a previous study, we found no apparent changes in ellipticity upon addition of the substrates to native MS.40 Adding AcCoA to demetallized MS prior to Mg2+ or glyoxylate yielded no change in fluorescence intensity or λmax (Figure S5B). However, when a saturating concentration of AcCoA was added after Mg2+ and glyoxylate, the spectrum recovered to that of the Mg2+-bound spectrum. This is consistent with a sequential reaction mechanism in which glyoxylate binding is a prerequisite for acetyl-CoA binding, and that Mg2+ remains bound in the active site upon release of the reaction products.19,26−29,34,38−40 The enhancement in MS tryptophan fluorescence following addition of Mg2+ suggested that this spectroscopic signal might be used quantitatively to measure binding of Mg2+ to the enzyme. PA MS tryptophan fluorescence increased hyperbolically with an increase in Mg2+ concentration, as is typical of a saturable binding system (Figure 6A). In the presence of glyoxylate, the same Mg2+ titration also yielded a binding isotherm, except that increasing Mg2+ concentrations led to saturable quenching of fluorescence (Figure 6A). Scatchard analysis of the fluorescence data yielded apparent Kd values for the Mg2+−MS complex of 525 and 653 μM in the absence and

Figure 6. (A) Mg2+ binding curves in the presence (red) and absence (blue) of 1 mM glyoxylate. Note that glyoxylate quenches the tryptophan fluorescence of P. aeruginosa malate synthase G in the presence of Mg2+. Error bars correspond to ±1 standard deviation. (B) Scatchard analysis of Mg2+ binding data. F


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Biochemistry Table 3. SiteMap Parameters for Predicted Binding Sites A−E site site site site site site

A B C D E

SiteScore

size (no. of site points)

Dscore

volume (Å3)

hydrophobic

hydrophilic

1.10 0.99 0.97 0.96 0.87

123 188 163 110 80

1.16 1.02 1.02 0.97 0.87

475.01 569.03 461.24 269.12 156.53

1.60 0.58 0.59 0.50 0.79

0.72 0.95 0.89 1.06 1.04

Figure 7. (A) Colored mesh represents the possible binding pockets predicted by SiteMap in P. aeruginosa malate synthase G (represented as a gray cartoon): site A, yellow; site B, orange; site C, magenta; site D, blue; site E, cyan. Site A is located between a β-hairpin (amino acids 622−628) in domain II and α-helices in the TIM barrel. Site B is composed of loops in the TIM barrel and domains II and III. Site C is located on the edge of the protein between helix α1 in the N-terminal region and the final α-helix of the protein at the C-terminus, along with a β-hairpin (amino acids 345− 350) from the TIM barrel. Site D is composed of a loop in the N-terminal region and α-helices in the TIM barrel. Site E sits near the loops of the Nterminus and C-terminus, and a flexible loop in the TIM barrel. (B) Site A is a hydrophobic pocket, and the binding site residues are shown as sticks. (C) The polar and hydrophobic residues of site B are shown as sticks. These include the Arg340 and Asp631 amino acid side chains involved in catalysis and the active site fluorophore, Trp539.

overlaps the substrate binding pocket, surrounding the βhairpin of domain II (amino acids 615−631), which borders the active site. Because of the structural conservation and functional significance of sites A and B, we looked for published inhibitors of MS in other organisms that might bind in these sites. To date, however, the few known inhibitors of MS solely target the active site and are either glyoxylate analogues, Mg2+ chelators, or endogenous metabolic intermediates.2,15,26,29,34−36 An example of such Mg2+ chelators is a class of compounds, phenyldiketoacids (PDKA), that was discovered to inhibit Mtb MS.4,35,36 Mtb MS complexed with the PDKA inhibitors did not show significant conformational changes; however, as seen for ethylene glycol (EDO) in this work, the 1,2-diol moiety in these inhibitors acts as a bidentate ligand of the Mg2+ in the Mtb MS active site. Another example is a study in which a highthroughput screen against PA grown on acetate identified small molecule inhibitors of MS.15 In silico binding of these hits following an induced-fit docking procedure to homology

models of an Mtb MS crystal structure alluded to a likely interaction with the active site Mg2+. In a different study of Mtb MS complexed with chemical fragments, 18 fragments that formed a hydrogen bond with the backbone carbonyl of Met631 in the active site were identified.35 Met631 (Met629 in PA MS) is conserved in the MS superfamily and is found in site A. The scarcity of PA MS inhibitors highlights the potential of targeting it through alternative binding pockets. Computational Analysis of Structural Waters. The solvation network in a crystal structure plays an important role in determining protein−ligand binding affinities. Our PA MS crystal structure incorporates 691 water molecules per molecule of MS, as opposed to 450 water molecules per molecule of Mtb MS (PDB entry 1N8I). However, hydration networks are dynamic, so we used the molecular dynamic simulator, WaterMap, to better understand the possible impact of water molecules on ligand binding. The location of the predicted stable hydration sites was consistent with the observed G


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Figure 8. Alignment of malate synthase G crystal structures (gray, P. aeruginosa; green, E. coli; cyan, M. tuberculosis; orange, M. leprae). Predicted binding site A is shown in orange mesh with substrates glyoxylate and AcCoA (shown as sticks).

(Figure 10A) revealed cavity regions along with unstable hydration sites in sites A (Figure 10B) and B (Figure 10C). The thermodynamic parameters associated with the hydration sites shown in panels B and C of Figure 10 are provided in Table S1. Site B has fewer unstable hydration sites than site A does, which supports the hydrophilic nature of the pocket. In contrast, site A is more hydrophobic. However, the unstable hydration sites in site A are not continuous, suggesting that a large molecule with several hydrophobic regions would be required to fit in neatly. One particularly interesting cavity is located near the AcCoA binding site (blue mesh in Figure 10C). This cavity is seen to bind a hydrophobic small molecule [indole-3-carboxylic acid (ICO)] in Mtb MS (PDB entry 5CC5). In that structure, ICO occupies the space between the glyoxylate and AcCoA binding sites (Figure 11), corresponding to the exact cavity identified here in PA MS.

crystallographic waters (a snapshot of the active site water molecules is shown in Figure 9).

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Figure 9. Hydration sites (brown spheres representing unstable water molecules and green spheres representing stable water molecules) predicted by WaterMap superposed with malate synthase G crystal waters (cyan spheres). Catalytic amino acid residues (Arg340, Glu432, and Asp631) are shown as sticks.

CONCLUSIONS

The need for new antibacterial agents against P. aeruginosa is now critical. Because of the importance of the glyoxylate shunt for persistence in infection scenarios, we have characterized the structure and properties of P. aeruginosa malate synthase G, with a special emphasis on identifying potential drug binding sites. We report the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. We used the crystal structure to influence experimentation, and we show that events in the enzyme active site can be monitored non-invasively using intrinsic tryptophan fluorescence as a probe. Our computational data reveal two sites in the structure that are promising for drug development as well as the thermodynamic properties of hydration sites within the predicted binding sites. Our findings contribute to the growing body of evidence that malate synthase G is a tractable drug target and provide a means for inhibitor development against P. aeruginosa using structure-based approaches.

The hydration sites predicted by WaterMap gave additional insights into the thermodynamics of the solvation network (Figure 10). The water molecules in the hydration sites colored green are high-energy water molecules. Typically, these water molecules are favorable for binding, and ligands that displace these water molecules must compensate for this free energy loss. Thus, it is better to avoid these sites during ligand design. Conversely, hydration sites colored red/brown portray lowerenergy sites or unfavorable sites. Thus, replacing these water molecules should favor an increase in free energy. WaterMap also predicts cavity regions that are devoid of water molecules, so targeting these regions may help in the search for highaffinity binders. The WaterMap analysis confirmed the top-ranked predicted binding sites, A and B (Table 3), as druggable. WaterMap H


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Biochemistry

Figure 10. (A) Hydration sites of P. aeruginosa malate synthase G along with the cavity map (blue mesh) predicted by WaterMap. The relative free energy of the water molecules is depicted by a green to red color gradient, as indicated. Predicted binding sites A and B are outlined in yellow and brown, respectively. (B) Site A. Yellow surface and active site residues within the site are shown as sticks, and blue labels indicate hydration site numbers. (C) Site B. An orange surface and cavity map (blue mesh) partially occupy the site. The hydrophobic amino acids are shown as sticks, and the hydration sites are labeled in blue.

(Figure S2), size exclusion chromatography column elution profile of MS (Figure S3), Ramachandran plot (Figure S4), supplemental fluorescence data (Figure S5), CD data and CD methods (Figure S6), the general catalytic reaction scheme of MS (Figure S7), and hydration site thermodynamics (Table S1) (PDF) Accession Codes

The atomic coordinates and structure factors obtained in this work have been deposited in the PDB as entry 5OAS.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +44 (0)1223 333653. ORCID

Alyssa C. McVey: 0000-0001-9284-7652 David R. Spring: 0000-0001-7355-2824 Taufiq Rahman: 0000-0003-3830-5160

Figure 11. Cavity map predicted by WaterMap (blue mesh). Indole-3carboxylic acid (ICO), which partially occupies the cavity region in M. tuberculosis malate synthase G (PDB entry 5CC5), is shown as green sticks. Substrates glyoxylate (teal sticks) and AcCoA (cyan sticks) from PDB entry 1P7T are also presented to illustrate the binding location.

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Author Contributions

M.W. conceived the study. A.C.M., P.M., X.C., and S.B. performed experimental work. All authors contributed to the manuscript and have approved the final version.

ASSOCIATED CONTENT

S Supporting Information *

Funding

This work was supported by the European Commission’s Horizon 2020 Grant 642620 to M.W. and A.P. and BBSRC Grant BB/M019411/1 to M.W.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00852. Evolutionary tree based on sequence identity of other MSs (Figure S1), sequence alignment of other MSs

Notes

The authors declare no competing financial interest. I


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ACKNOWLEDGMENTS We thank the Diamond Light Source for access to beamline I03 (proposal MX-14043). We are grateful to Dr. Dima Chirgdaze and Dr. Paul Brear (University of Cambridge) for their assistance with X-ray data collection and analysis.

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ABBREVIATIONS AcCoA, acetyl-coenzyme A; CoA, coenzyme A; DTNB, 5,5dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GlcB, malate synthase G gene; GST, glutathione S-transferase; ICL, isocitrate lyase; ICO, indole-3-carboxylic acid; IPTG, isopropyl β-D-thiogalactopyranoside; MS, malate synthase G; Mtb, M. tuberculosis; ORF, open reading frame; PA, P. aeruginosa; PDKA, phenyldiketoacid(s); PDB, Protein Data Bank; PEG, polyethylene glycol; rmsd, root-mean-square deviation; TCEP, tris(2carboxyethyl)phosphine; Tris, tris(hydroxymethyl)aminomethane.

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